Thymidylate synthase inhibitors are a class of drugs that are widely used for the treatment of various cancers. One effect of these compounds is to increase the levels of uracil bases that are incorporated into newly synthesized genomic DNA. Excessive uracil incorporation into DNA can become toxic to cells, and this is believed to contribute to the ability of thymidylate synthase inhibitors to target rapidly dividing cancer cell. The primary DNA repair enzyme that initiates the base excision repair pathway to correct these uracil lesions in genomic DNA is called the nuclear uracil DNA glycosylase (UNG2). We propose a relationship between the uracil excision activity of UNG2 and the efficacy of 5-fluorouracil, a prototypical thymidylate synthase inhibitor. The activity of UNG2 is regulated in part by its localization within the nucleus and its accessibility to sites of DNA damage. Interactions of UNG2 with proliferating cell nuclear antigen (PCNA) and replication protein A (RPA) can affect its localization, specifically to the DNA replication fork; therefore, we reason that its ability to excise uracil from newly synthesized DNA during 5-fluorouracil treatment is als dependent on these protein- protein interactions. Specific UNG2 residues within the PCNA and RPA binding domains can be post- translationally modified, and we hypothesize that these post-translational modifications (PTMs) affect UNG2 interactions with these proteins. Post-translational modification of UNG2 is therefore hypothesized to modulate the susceptibility of cancer cells to 5-fluorouracil toxicity by affecting protein-protein interactions and subsequently removal of uracil from DNA. To test our hypotheses, we will use state-of-the-art protein semi- synthesis methods to generate UNG2 variants that have PTMs incorporated at specific residues. The post- translationally modified UNG2 variants will be used in biochemical assays that quantify their activity and their affinities for PCNA and RPA. The structural basis for modulating UNG2 interactions with PCNA and RPA will also be determined. To confirm that PTMs affect protein-protein interactions and the localization of UNG2 in the nucleus, we will use live cell fluorescence imaging of microinjected semi-synthetic UNG2 variants. Finally, the efficacy of 5-fluorouracil will be examined in cells that are transfected with UNG2 mutants that have altered affinities for PCNA and/or RPA. Together, the results of this interdisciplinary projec will address the contribution of a specific set of UNG2 PTMs towards the efficacy of 5-fluorouracil and other thymidylate synthase inhibitors.